WO2024051687A1 - Topical formulation containing pirfenidone and use thereof - Google Patents

Abstract

A pirfenidone topical formulation is provided, comprising a therapeutically effective amount of pirfenidone, water, and at least one cosolvent, the cosolvent being selected from a combination of dimethyl sulfoxide and an alcohol solvent. The formulation exhibits high skin permeability, good stability, no irritation, excellent skin tolerance, and ease of use. The formulation can be used in the preparation of drugs for preventing or treating diseases related to excessive proliferation of skin or connective tissues and chronic skin injuries.

Description

A kind of topical preparation containing pirfenidone and its use

This application claims priority to the following earlier applications: The patent application number submitted to the China State Intellectual Property Office on September 6, 2022 is 202211086265.9, and the invention is titled "A topical preparation containing pirfenidone and its use" Apply first. The entirety of said prior application is incorporated into this application by reference.

Technical field

The invention belongs to the field of pharmaceutical preparations, and specifically relates to a topical preparation containing pirfenidone and its use.

Background technique

Pirfenidone, chemically named 5-methyl-1-phenyl-2-(1H)pyridone, is a compound with broad-spectrum anti-fibrotic effects that can prevent and reverse fibrosis and scarring The formation of , its chemical structure is as follows:

In 2014, pirfenidone was approved by the U.S. Food and Drug Administration (FDA) for marketing under the trade name Pirfenidone has become the first drug approved in multiple countries for the treatment of idiopathic pulmonary fibrosis (IPF).

In recent years, pirfenidone has been extensively studied for other uses. For example, WO00/16775 discloses the use of pirfenidone for the treatment and prevention of skin lesions, especially lesions of a fibrotic nature, such as fibrotic damaged tissue, contact infectious warts, contact dermatitis, burns and scars. WO99/47140 discloses the use of 2-1H-pyridone compounds, especially pirfenidone, as a local disinfection treatment, which can be used to treat fungi, bacteria, etc. on the skin surface. WO01/62253 discloses the use of pirfenidone in the treatment of epilepsy. US Patent No. 4,052,509 discloses the use of pirfenidone as an anti-inflammatory agent. Mexican Patent 182,266 discloses that pirfenidone has antifibrotic activity. Chinese patent document CN111246840A discloses that pirfenidone can be used to prevent or treat chronic skin injuries and injuries caused by neuropathic ulcers, especially for the treatment of diabetic foot syndrome and the treatment of vascular ulcers.

In summary, many studies have shown that pirfenidone can be used to treat and prevent skin lesions such as abnormal skin scars and other defects caused by fibrosis or inflammation caused by surgical wounds, burns, accidents, ultraviolet rays, pregnancy, or chemical agents, but pirfenidone Nicotine has poor solubility in water, making it difficult to prepare stable external preparations.

CN10808641A discloses a gel composition containing pirfenidone. This application discloses the use of solubilizers such as NMP, ethanol and propylene glycol to prepare the gel preparation, which can be used for tissue recovery with fibrotic lesions and for fibrosis. Prevention of lesions, but its stability, transdermal effect and in vivo effect have not been studied.

Therefore, there is a need to further provide a pirfenidone drug combination with high stability, good transdermal effect and efficacy. things.

Contents of the invention

One object of the present invention is to provide a topical preparation of pirfenidone, which has high skin permeability, good stability, low irritation, can efficiently penetrate the skin and take effect safely.

In order to achieve the above objects, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a pirfenidone topical formulation, which includes a therapeutically effective amount of pirfenidone, water and at least one co-solvent.

In some embodiments, the co-solvent is selected from dimethyl sulfoxide (DMSO) and an alcoholic solvent, and the alcoholic solvent is selected from one of ethanol, propylene glycol, isopropyl alcohol, 1-propanol, and butylene glycol. species, two or more species. Preferably, the co-solvent is a combination of DMSO and ethanol, or a combination of DMSO and propylene glycol, or a combination of DMSO, ethanol and propylene glycol.

In some embodiments, the amount of water used in the formulation is 10 wt% to 55 wt%, preferably 15 wt% to 50 wt%, such as 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt% or 45 wt%.

In some embodiments, when the co-solvent is a combination of DMSO and ethanol, the formulation contains 15 to 35 wt% DMSO, 15 to 30 wt% ethanol, based on the total weight of the topical formulation; preferably, the formulation The formulation contains 15 to 25 wt% DMSO, 15 to 25 wt% ethanol, for example 20 wt% DMSO and 20 wt% ethanol, or 20 wt% DMSO and 25 wt% ethanol.

In some embodiments, when the co-solvent is a combination of DMSO, ethanol, and propylene glycol, the formulation includes 15 to 35 wt% DMSO, 15 to 30 wt% ethanol, 15 wt% based on the total weight of the topical formulation. to 25 wt% propylene glycol; preferably, the formulation contains 15 to 25 wt% DMSO, 15 to 25 wt% ethanol, and 20 to 25 wt% propylene glycol.

In some embodiments, pirfenidone is present in an amount of 1 to 14 wt%; preferably, 4 to 12 wt%, based on the total weight of the pirfenidone topical formulation; For example, it exists in an amount of 6 to 12 wt%, for example, it exists in an amount of 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11% wt, or any interval range thereof.

In some embodiments, the topical pirfenidone formulations of the present invention can be formulated into different dosage forms, such as solutions, suspensions, creams, ointments, lotions, and gels. Preferably, the topical pirfenidone preparation of the present invention is a solution or gel. More preferably, the topical pirfenidone preparation of the present invention is a gel.

In some embodiments, optionally, the pirfenidone topical formulation further comprises a gelling agent. The gelling agent may be one known in the art for use in topical formulations, including but not limited to: cellulose derivatives, polyvinylpyrrolidone, carbomer polymers, carbomer derivatives, malt One, two or more of dextrin, polydextrose, glucose binding agent, polyacrylamide gelling agent, carboxypolymethylene, polyvinyl alcohol, poloxamer, and polyethylene glycol; preferably Preferably, the gelling agent is selected from one, two or more types of cellulose derivatives, carbomer polymers, and carbomer derivatives. For example, the gelling agent is HPC, carbomer 980, carbomer 981, etc.

In some embodiments, the gelling agent is present in an amount from 0.05% to 5% by weight, preferably from 0.1% to 2% by weight, such as from 0.3% to 1% by weight, based on the total weight of the pirfenidone topical formulation. Quantity exists.

In some embodiments, optionally, the pirfenidone topical formulation further includes a humectant, which may be a humectant known in the art for topical formulations, including but not limited to: One, two or more of glycerin, urea, allantoin, sodium hyaluronate, polyethylene glycol, squalane, cyclomethicone. Preferably, the moisturizing agent is selected from one, two or three types of glycerin, allantoin, and sodium hyaluronate.

In some embodiments, the humectant is present in an amount from 0.5 to 10 wt%, preferably from 2 to 8 wt%, such as from 3 to 6 wt%, based on the total weight of the pirfenidone topical formulation.

In some embodiments, optionally, the pirfenidone topical formulation includes a pH adjuster, which may be a pH adjuster known in the art for topical formulations, particularly a base. The pH adjuster is, for example, one, two or three types selected from triethanolamine, sodium hydroxide, and triethylamine; preferably, the pH adjuster is triethanolamine. The pH adjuster is used in an appropriate amount, and it is appropriate to adjust the pH of the topical preparation to a range of 5.0 to 7.5, and more preferably to a pH of 5.5 to 7.5.

In some embodiments, optionally, the pirfenidone topical formulation further includes a preservative, which may be a preservative known in the art for topical formulations, for example, selected from p-hydroxyl One, two or more of methyl benzoate (methyl hydroxybenzoate), propyl parahydroxybenzoate (propyl parahydroxybenzoate), benzyl alcohol, benzoic acid, and phenol.

In some embodiments, the preservative is present in an amount from 0.01% to 2% by weight, such as from 0.05% to 1% by weight, based on the total weight of the pirfenidone topical formulation.

In some embodiments, optionally, the pirfenidone topical preparation of the present invention further includes one, two or more other pharmaceutically acceptable auxiliary materials, including but not limited to: antioxidants, thickeners , stabilizers, chelating agents, oily materials, emulsifiers, penetration accelerators, antimicrobial agents, sunscreen agents, fragrances, colorants, surfactants, etc. The excipients can be used in conventional amounts known in the art.

For example, the antioxidant may be one known in the art for use in topical formulations, such as selected from the group consisting of butylated hydroxyanisole (BHA), butylated hydroxytoluene, vitamin C, vitamin E, vitamin A, phylloside Vitamin, lycopene, retinyl palmitate, potassium metabisulfite, sodium metabisulfite, sodium thiosulfate pentahydrate, 3,4-dihydroxybenzoic acid, propyl gallate, alpha-lipoic acid , one, two or more of ascorbyl palmitate, sodium metabisulfite, ubiquinone, and selenium. For example, an antioxidant is butylated hydroxyanisole (BHA). The antioxidant may be present in an amount from 0.05% to 5% by weight, for example from 0.1% to 3% by weight, based on the total weight of the pirfenidone topical formulation.

For example, the thickening agent may be, for example, cellulose derivatives, polyvinylpyrrolidone, carbomer polymers, carbomer derivatives, maltodextrin, polydextrose, dextrate, carboxypolymethylene , one, two or more of polyvinyl alcohol, poloxamer and polyethylene glycol; more preferably, the thickener is hydroxypropyl cellulose. The thickening agent may be present in an amount of 0.05 to 5 wt%, preferably 0.1 to 4 wt%, such as 1 to 3 wt%, based on the total weight of the pirfenidone topical formulation.

In an exemplary embodiment, the pirfenidone topical formulation of the present invention comprises a therapeutically effective amount of pirfenidone, water and at least one co-solvent comprising DMSO and ethanol, wherein Based on the total weight of the formulation, the formulation includes 4 to 12 wt% pirfenidone, 15 to 30 wt% DMSO, and 15 to 30 wt% ethanol. Optionally, the formulation further contains 0.05wt% to 5wt% gelling agent (for example, selected from carbomer 980, carbomer 981 one or two). Optionally, the preparation further contains 0.05 wt% to 8 wt% of a moisturizing agent (for example, one, two or three selected from the group consisting of glycerin, allantoin, and sodium hyaluronate). Optionally, the formulation further contains 0.05% to 2% by weight of a preservative. Optionally, the preparation further contains a pH adjuster (for example, one or two selected from triethanolamine and NaOH) to adjust the pH range of the preparation to 5.0-7.5. Optionally, the preparation further contains other pharmaceutically acceptable auxiliary materials.

In an exemplary embodiment, the pirfenidone topical formulation of the present invention comprises a therapeutically effective amount of pirfenidone, water and at least one co-solvent comprising DMSO and ethanol, wherein Based on the total weight of the formulation, the formulation includes 6 to 12 wt% pirfenidone, 20 to 30 wt% DMSO, and 20 to 30 wt% ethanol. Optionally, the preparation further contains 0.4 wt% to 1 wt% of a gelling agent (for example, one or two selected from carbomer 980, carbomer 981). Optionally, the preparation further contains 3 to 6 wt% of a moisturizing agent (for example, one, two or three selected from the group consisting of glycerol, allantoin, and sodium hyaluronate). Optionally, the formulation further contains 0.05 to 0.5 wt% of a preservative. Optionally, the preparation further contains a pH adjuster (for example, one or two selected from triethanolamine and NaOH) to adjust the pH range of the preparation to 5.0-7.5. Optionally, the preparation further contains other pharmaceutically acceptable auxiliary materials.

In an exemplary embodiment, the formulation includes 4 to 12 wt% pirfenidone, 15 to 35 wt% DMSO, 15 to 30 wt% ethanol, 20 to 50 wt% water, 0.3 wt % to 1wt% gelling agent, 2wt% to 8wt% humectant, and an appropriate amount of pH adjuster to adjust the pH range of the preparation to 5.0-7.5.

In an exemplary embodiment, the formulation includes 4 to 12 wt% pirfenidone, 20 to 25 wt% DMSO, 20 to 25 wt% ethanol, 30 to 45 wt% water, 0.3 wt % to 1wt% gelling agent, 2wt% to 8wt% humectant, 0.05wt% to 1wt% preservative, and an appropriate amount of pH adjuster to adjust the pH range of the preparation to 5.0-7.5.

In a second aspect, the present invention also provides the use of the above-mentioned pirfenidone topical preparation in the preparation of medicaments for preventing or treating diseases related to skin or connective tissue hyperproliferation and chronic skin damage.

In a third aspect, the present invention provides a method for preventing or treating skin or connective tissue hyperproliferation-related diseases and chronic skin damage in a mammal (e.g., a human), the method comprising administering a therapeutically effective amount to the mammal (e.g., a human). The topical pirfenidone preparation of the present invention.

In some embodiments of the present invention, the diseases related to skin or connective tissue hyperproliferation include, but are not limited to, pathological scars (such as keloids, hypertrophic scars), acne scars, skin neoplastic fibrosis and cicatricial alopecia; the Chronic skin lesions include, but are not limited to, vasculitis, burn healing, diabetic foot syndrome, vascular ulcers, scleroderma, Peyronie's syndrome, Deutsch syndrome, or bursitis.

The inventor of the present invention unexpectedly discovered that when DMSO and alcohol solvents are used as co-solvents in pirfenidone topical preparations, its medicinal properties can be significantly improved. The resulting preparation overcomes the problem of low solubility of pirfenidone in water and skin problems. Technical problems such as low transmittance and sensitivity to skin irritation. The preparation has high skin permeability, good stability, non-irritation, good skin tolerance, and is easy to use. It can significantly improve the compliance of patients with keloids, pathological scars or hypertrophic scars, and can be developed into a very Promising topical agents.

Description of the drawings

Figures 1 to 4 respectively show the HE stained pathological sections of four different groups of rabbit ear surface scar areas 28 days after administration in the pirfenidone in vivo efficacy test of Test Example 4 of the present invention. The small figures in each figure Figures 1, 2, 3, and 4 respectively show the HE stained pathological sections of 4 samples in each group.

Definitions and explanations of terms

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entirety.

As used herein, the terms "includes," "includes," "has," "contains," or "involving" and other variations thereof herein are inclusive or open-ended and do not exclude others that are not. Enumerated elements or method steps.

The term "prevention or treatment" means the administration of a formulation of the invention to prevent, ameliorate or eliminate a disease or one or more symptoms associated with said disease, and includes: (i) prevention of a disease or disease state in lactation occur in animals, especially when such mammals are susceptible to the disease state but have not been diagnosed as suffering from the disease state; (ii) inhibit the disease or disease state, that is, arrest its progression; (iii) alleviate the disease or disease state, even if the disease or disease state resolves.

The term "therapeutically effective amount" means (i) treating or preventing a specified disease, condition, or disorder, (ii) alleviating, ameliorating, or eliminating one or more symptoms of a specified disease, condition, or disorder, or (iii) preventing or delaying An amount of a formulation of the invention that is associated with the onset of one or more symptoms of a particular disease, condition or disorder described herein. The amount of a formulation of the invention that constitutes a "therapeutically effective amount" will vary depending on the formulation, the disease state and its severity, the mode of administration and the age of the mammal to be treated, but can be routinely determined by one skilled in the art. based on its own knowledge and the contents of this disclosure.

The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue without multiple toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable excipients" refers to those excipients that have no obvious irritating effect on the organism and do not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, water, other solvents, etc.

As used herein, the term "solvent" is water.

As used herein, the term "co-solvent" refers to a substance that acts as a co-solvent in combination with other substances, such as solvent water.

As used herein, the term "chronic skin injury" refers to exuberant activity during various stages of wound healing that results in abnormal fibrotic tissue formation (eg, scarring).

As used herein, the term "diabetic foot syndrome." Diabetic foot is a clinical entity that refers to the presence of chronic hyperglycemia in patients with diabetes due to extrinsic and intrinsic factors such as trauma, local hygiene, and skeletal deformities. A syndrome resulting from the interaction of predisposing factors such as vasculopathy, neuropathy, and infection. Common typical clinical symptoms include lower limb ulcers and foot ulcers (also known as diabetic foot ulcers).

As used herein, the term "polyacrylamide gelling agent" primarily refers to the polyacrylamide gelling agent produced by SEPPIC under the name Sepineo (or Simulgel 600 ) sold as a mixture Sodium Acryldimethyl Taurate Copolymer/Isohexadecane/Polysorbate 80, a mixture Polyacrylamide/Isoparaffin C13-14/Laureth-7 (as sold by SEPPIC by name Products sold by 305.

Detailed ways

The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention. All technologies implemented based on the above contents of the present invention are covered by the scope of protection intended by the present invention. Experimental methods that do not indicate specific conditions in the examples are usually operated according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise stated, the percentages and parts used in the examples are by weight. The raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

The following abbreviations are used in the present invention: DMSO represents dimethyl sulfoxide; NMP represents N-methylpyrrolidone; Solutol HS 15 represents polyethylene glycol (PEG)-15 hydroxystearate; Transcutol represents diethylene glycol Monoethyl ether; HPC stands for hydroxypropyl cellulose and BHA stands for butyl hydroxyanisole. "-" in the table below means that the corresponding ingredient is not included or has not been added.

Example 1 Solubility test of pirfenidone

This example examines the solubility of pirfenidone in water/DMSO/alcohol solvents.

Experimental method: Add excess pirfenidone to the solvent system listed in Table 1. The resulting suspension was stirred overnight and filtered through a 0.22 μm filter. Dilute the filtrate to the appropriate concentration. The concentration of pirfenidone in each solution was determined by HPLC method. The solubility results of pirfenidone in different solvent systems are shown in Table 1.

Table 1. Solubility of pirfenidone in different solvent systems

It can be seen from the experimental results that although purified water is a common solvent in topical preparations, pirfenidone in water The solubility is low. Even if DMSO is added for solubilization, the solubility is difficult to reach the desired concentration (80 mg/mL) in topical preparations. However, if ethanol or propylene glycol is further added for solubilization, the solubility of pirfenidone in water can be significantly improved. For example, pirfenidone has good solubility in water/DMSO/ethanol or water/DMSO/propylene glycol, and can reach the desired concentration of pirfenidone in topical formulations (80 mg/mL).

Example 2 Compatibility test of raw materials and excipients of pirfenidone topical preparation

In this embodiment, a compatibility test of raw materials and excipients was conducted to examine the effects of excipients such as cosolvents, gelling agents, and moisturizers on the stability of pirfenidone topical preparations. Gelling agents (eg, carbomer) and humectants (eg, glycerin, allantoin, sodium hyaluronate) are commonly used excipients in topical formulations.

Experimental method: Mix pirfenidone and excipients (including cosolvents, gelling agents, moisturizers, etc.) in a weight ratio of 1:99 (API pirfenidone accounts for 1% by weight, and the total weight of other excipients (99%), place it under high temperature (60°C, dry) conditions for 30 days, and test its total degradation impurity content and maximum single impurity content. The experimental results are shown in Table 2.

Table 2. Compatibility test of raw materials and excipients

Experimental results show that pirfenidone has excellent properties among the co-solvents (DMSO, ethanol), commonly used gelling agents (carbopol), and humectants (glycerin, allantoin, sodium hyaluronate) tested in Example 1. Therefore, these excipients have good compatibility with pirfenidone and can be used in the pirfenidone topical preparation of the present invention.

In addition, this application also tested the stability of pirfenidone in the presence of Transcuto. Although Transcutol can also solubilize pirfenidone in a DMSO + water system, the use of Transcutol results in product instability. For example, a preparation containing pirfenidone/Transcutol/water at a ratio of 1%:23.5%:75.5% will produce obvious degradation impurities after being placed under high temperature (60°C, dry) conditions for 30 days. It is speculated that Transcutol may induce pirfenidone. Degradation of nitone.

In summary, it can be seen that not any organic solvent can be used as a compatibilizer in the pirfenidone topical preparation of the present application. The DMSO+alcohol solvent used in the present invention not only can solubilize, but also does not affect the stability of pirfenidone and is a good co-solvent.

Example 3 Preparation of pirfenidone topical preparation

In this example, a topical preparation of pirfenidone was prepared according to the prescription shown in Table 3.

Preparation method: According to the prescription, 1) mix pirfenidone and co-solvent; 2) mix the gelling agent with an appropriate amount of purified water; 3) mix the humectant with the remaining amount of purified water; 4) mix step 1) , 2), 3) The mixture obtained is mixed evenly, and a pH adjuster is added to adjust the pH to 5.5-7.5, and stirred until uniform.

Table 3. Topical formulations of pirfenidone

Example 4 Preparation of pirfenidone topical preparation

In this example, a topical preparation of pirfenidone was prepared according to the prescription shown in Table 4.

Preparation method: According to the prescription, 1) mix pirfenidone and cosolvent, add preservative and mix; 2) mix gelling agent and Mix an appropriate amount of purified water; 3) Mix the moisturizer with the remaining amount of purified water; 4) Mix the mixture obtained in steps 1), 2), and 3) evenly, add a pH adjuster to adjust the pH to 5.5~7.5, and stir until Uniform, that's it.

Table 4. Topical formulations of pirfenidone

Example 5 Preparation of pirfenidone topical preparation

In this example, a topical preparation of pirfenidone was prepared according to the prescription shown in Table 5.

Preparation method: Refer to Example 4.

Table 5. Topical formulations of pirfenidone

Example 6 Preparation of pirfenidone topical preparation

In this example, a topical preparation of pirfenidone was prepared according to the prescription shown in Table 6.

Preparation method: According to the prescription, 1) mix pirfenidone and co-solvent, add antioxidant and mix well; 2) mix gelling agent and purified water; 3) mix the mixture obtained in steps 1) and 2) evenly, Add pH adjuster to adjust pH to 5.5~7.5, stir until uniform, and it is ready.

Table 6. Topical formulations of pirfenidone

Example 7 Preparation of pirfenidone topical preparation

In this example, a topical preparation of pirfenidone was prepared according to the prescription shown in Table 7.

Preparation method: Refer to Example 3.

Table 7. Pirfenidone topical formulations

Example 8 Preparation of pirfenidone topical preparation

In this example, a topical preparation of pirfenidone was prepared according to the prescription shown in Table 8. The control preparation prescription C1 was prepared with reference to the prescription of KitosCell, a pirfenidone gel preparation marketed in Mexico.

Preparation method: According to the prescription, 1) mix pirfenidone and co-solvent, add preservatives (if any) and other auxiliary materials to mix; 2) mix the gelling agent with an appropriate amount of purified water; 3) mix step 1) , 2) Mix the resulting mixture evenly, add a pH adjuster to adjust the pH to 5.5-7.5, and stir until uniform.

Table 8. Topical formulations of pirfenidone

Test Example 1 Stability of pirfenidone topical preparation

This test example examines the stability of pirfenidone topical preparations.

Test method: The pirfenidone topical preparations in Examples 3 to 8 were subjected to high temperature (60°C, drying) and accelerated conditions. Stability research experiments were conducted under the conditions of (40°C ± 2°C, 75% RH ± 5% RH) to examine the physical and chemical stability of each preparation at 0 days and 30 days. The experimental results are shown in Table 9 below.

Table 9. Stability study of pirfenidone topical formulations



Note: The 0-day measurement data is the measurement value obtained after the sample preparation is completed.

Experimental results show that when the pirfenidone topical preparation of the present invention is placed under high temperature and accelerated conditions for 30 days, the color properties do not change, the impurity content does not increase significantly, and all maintain good stability. Among them, the viscosity of different formulations varies greatly, which is caused by the different viscosity of the gelling agent. For example, the viscosity of carbomer 980 with a concentration of 0.5% w/w is 40,000 to 60,000 centipoise at pH 7.5, which is viscous and has weak fluidity; while carbomer 981 with a concentration of 0.5% w/w is The viscosity at pH 7.5 is 4,000 to 10,000 centipoise, and the fluidity is relatively strong. Therefore, topical gel preparations prepared using different gelling agents have different viscosities. When the preparation is applied, different viscosities can produce different feelings, and all can be made into medicines without affecting the stability and efficacy of the preparation. In addition, the results show that the addition of humectants, preservatives, antioxidants, etc. of the present invention will not affect the stability of the preparation, which is consistent with the raw material compatibility test results of Example 2.

In the formulation of the present invention, the co-solvent can be a combination of DMSO and alcohol solvents. In comparison, the control prescription C1 uses polyethylene glycol glyceryl hydroxystearate and NMP as solubilizers or co-solvents. The gel preparation becomes darker and the total degradation products increase after being placed at 60°C for 30 days. to 0.34%, the stability of the preparation is poor. This may be due to the influence of NMP and the preservatives diazoalkyl imidazuron and butyl iodopropynyl carbamate on the stability of the preparation; the control prescription C2 uses hydroxystearic acid Polyethylene glycol glyceride and propylene glycol are used as co-solvents, and their stability is better than the control prescription C1. In the control recipe C3, Transcutol and DMSO are used as co-solvents, and the total degradation impurities increased to 0.60% after being left at 60°C for 30 days. It is speculated that Transcutol is The stability of the preparation is affected, and this result is consistent with the raw material compatibility test results in Example 2. In summary, it can be seen that the topical pirfenidone preparation of the present invention has better physical and chemical stability.

Test Example 2 In vitro transdermal test of pirfenidone topical preparation

In this test example, an in vitro transdermal test was performed on the pirfenidone topical preparations in Examples 3 to 8 to evaluate the ability of each pirfenidone topical preparation to penetrate the skin layer. Generally, hairless guinea pigs or pigs are accepted models for predicting human skin absorption rates. In contrast, other models using experimental animals with hair, such as rats, rabbits, and guinea pigs, often lead to overestimation of human skin absorption rates.

Test method: Use the back skin of Bama Xiang pigs (1.5 months old, Linxi County Jingde Agricultural Products Sales Co., Ltd., No. 20220454, 20220455, 20220456) as the permeability membrane, and use a vertical diffusion cell to conduct skin penetration/retention tests. Take 0.8-1.0mm thick pig skin and cut off the hair on the skin. Confirm skin resistance to ensure skin integrity. The skin was placed on top of a diffusion cell in contact with the receptor, the receiving phase of which was filled with phosphate buffered saline (PBS) (pH 6.8). Approximately 25 mg of pirfenidone topical preparation was added to the skin surface in the donor compartment. After 24 hours, the receiving phase medium was taken, filtered with a 0.45 μm filter, and analyzed by high-performance liquid chromatography (HPLC) to determine and calculate the average formulation prescription Cumulative penetration amount, the results are shown in Table 10.

Table 10. 24h cumulative penetration of different formulations

The experimental results show that the 24h cumulative penetration amount of control prescription C1 is 405.7 μg/cm ² , the 24h cumulative penetration amount of control prescription C2 is only 173.0 μg/cm ² , and the 24h cumulative penetration amount of control preparation C3 is higher than that of control prescriptions C1 and C2. The 24-hour cumulative penetration amount of the pirfenidone topical preparation on the skin is about 800-1300 μg/cm ² , which is about 2-7 times that of the control prescription. In summary, the preparation of the present invention has a higher cumulative skin penetration, which may be the result of DMSO, ethanol and propylene glycol acting as co-solvents to promote the penetration of active pharmaceutical ingredients. In addition, experimental results show that the addition of humectants, preservatives and antioxidants will not affect the cumulative permeability of the preparation.

Test Example 3 Skin Irritation Test

In addition to the amount of penetration noted above, the irritation potential of topical formulations is also an important consideration. This test example examines the skin irritation of pirfenidone topical preparations through an acute skin irritation test (i.e., a single application test).

Test method: Select 15 volunteers as subjects and try different formulations shown in Table 14 in sequence. under test Test the drug on the back of the hand and the inside of the arm. After cleaning the back of the hand and the inside of the arm, select an area of about 2cmx2cm (not covered by heavy body hair), apply about 0.1g of the sample to the selected area, smooth it to a thin layer, 2- Carry out skin irritation score within 3 minutes. The higher the skin irritation score, the stronger the irritation of the corresponding preparation.

Scoring method: Refer to the "Technical Safety Specifications for Cosmetics" (2015 Edition) skin irritation/corrosion test method and set the following scoring method. Calculate the pain score according to formula 1 [skin pain score = (∑ hand pain score × 0.4 + ∑ inner arm pain score × 0.6)/number of subjects], and perform pain scoring according to the skin pain response scoring standards in Table 11; if there is pain Wipe off the test drug immediately after applying the test drug for 2-3 minutes. For samples without obvious tingling, wait 30 minutes before wiping off the test drug. After wiping off the test drug, observe the skin irritation reaction; and according to formula 2 [skin irritation score = (∑ hand erythema and edema score × 0.4 + ∑ inner arm erythema and edema score × 0.6)/number of subjects] Calculate the skin irritation score, and conduct skin irritation scoring according to the skin irritation reaction scoring standards in Table 12; and according to Formula 3 (Total skin irritation score = skin pain score + skin irritation score) Calculate the total skin irritation score, and evaluate the skin irritation intensity according to Table 13. The experimental results are shown in Table 14.

Table 11 Skin pain response scoring criteria

Table 12 Skin irritation reaction scoring criteria

Table 13 Skin irritation intensity evaluation table

Table 14 Skin irritation evaluation results of different prescription preparations

The experimental results showed that the subjects had obvious slight pain and obvious mild irritation during the application of the control preparation prescription C1, which may be caused by the large dosage of NMP; while the preparation of the present invention does not contain NMP, but uses Using an appropriate amount of DMSO with ethanol and/or propylene glycol as a co-solvent, the subjects basically felt no pain or irritation after application. Among them, prescriptions 5, 6 and 16 had the best experience. In addition, adding a humectant to the topical formulation of pirfenidone can also keep the skin moist and reduce irritation, especially when subjects need to apply the drug for a long time, helping to improve skin comfort.

Test Example 4 In vivo efficacy test of pirfenidone

Pathological scars often occur after damage to the deep dermis, causing aesthetic and functional damage to patients and seriously affecting their quality of life. Since human scar tissue is often limited in clinical sampling, most drug efficacy studies require the use of experimental animal models. Establishing a wound on a rabbit's ear and removing the perichondrium can slow down the shrinkage of the wound and slow its healing, eventually leading to scarring. This model is often used to study the in vivo efficacy of scar drugs. This experiment simulates clinical use, applying external preparations to rabbit ears that have formed wounds to treat scars, and observing scar healing through HE pathological sections.

Test materials: 4 groups of experimental preparations, of which group G1: preparation prescription 6 of the present invention; group G2: the matrix of preparation prescription 6 of the present invention, excluding pirfenidone; group G3: control prescription C1; group G4: model group , without applying any preparation).

Test instruments: dehydrator (DIAPATH, Donatello), embedding machine (Wuhan Junjie Electronics Co., Ltd., JB-P5), freezing table (Wuhan Junjie Electronics Co., Ltd., JB-L5), pathology microtome (Shanghai Leica Instruments Co., Ltd., RM2016), tissue spreader (Jinhua Kedi Instrument Equipment Co., Ltd., Zhejiang Province, KD-P), oven (Tianjin Lai Bo Rui Instrument Equipment Co., Ltd., GFL-230), anti-falling slides (Servicebio), Upright optical microscope (NIKON ECLIPSE E100, Nikon, Japan), imaging system (NIKON DS-U3, Nikon, Japan), xylene (Sinopharm Chemical Reagent Co., Ltd., 10023418), absolute ethanol (Sinopharm Chemical Reagent Co., Ltd., 100092683 ), HE dye solution set (Servicebio; G1003), central gum (Sinopharm Chemical Reagent Co., Ltd., 10004160), New Zealand rabbit (Leifu Breeding Farm, Pukou District, Nanjing, license number: SCXK (Su) 2019-0005).

Test method: 16 rabbit ears of 8 male New Zealand rabbits (1.7~2.0kg) were randomly divided into 4 groups, each group had 4 rabbit ears. After marking, they were adaptively raised for 1 week, and then the rabbits were anesthetized and shaved with a razor. Shave the ventral hair of rabbit ears and perform routine disinfection. Use a wound punch with a diameter of 1cm to measure the area on the ventral side of the rabbit ear. After touching the surface of the rabbit ear, press it tightly. Use a heavy object to tap the wound punch gently and turn it three or four times. Each rabbit ear will cause 4 full-thickness skin wounds of 1cm×1cm, with an interval of approximately 1.5cm between each wound. The skin in the wound area is removed together with the underlying perichondrium, leaving the cartilage intact. After the operation, the wound was disinfected with iodophor, and administration was started the next day. The administration method is shown in Table 15 below.

Table 15

After each administration, a dressing needs to be used to cover the administration site of the rabbit ear. Before each administration, a cotton swab dipped in physiological saline should be used to wipe the administration site. 28 days after administration, the rabbits were euthanized. The scar and surrounding normal tissue within 3 mm are cut and removed together with the cartilage. At the same time, a normal rabbit ear tissue was selected as a blank control. A landmark wound of each rabbit ear was selected and fixed with 10% formalin, sectioned and stained with HE.

The slicing steps are: fresh tissue is fixed in fixative for more than 24 hours. Remove the tissue from the fixative and use a scalpel to smooth the tissue at the target site in the fume hood. Place the trimmed tissue and corresponding labels in the dehydration box. Put the dehydration box into the dehydrator for dehydration in a gradient sequence: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, absolute ethanol I for 30 minutes, absolute ethanol II for 30 minutes, alcohol benzene (ethanol, Xylene mixed solvent, V _ethanol : V _xylene = 1:1) 5 to 10 minutes, xylene I 5 to 10 minutes, xylene II 5 to 10 minutes, melt paraffin I at 65°C for 1 hour, melt paraffin II at 65°C for 1 hour, 65°C Melt paraffin III for 1 h. The tissue soaked in wax is embedded in the embedding machine. First put the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and attach the corresponding label. Cool on a -20°C freezing platform. After the wax solidifies, remove the wax block from the embedding frame and trim the wax block. Place the trimmed wax block into a -20°C freezer to cool, and then place the cooled wax block into paraffin microtome slices with a thickness of 4 μm. The slices were floated on warm water at 40°C in a spreading machine to flatten the tissue. The tissue was picked up by a glass slide and baked in a 60°C oven. After the water-baked dry wax is baked, take it out and store it at room temperature for later use.

The steps of HE staining are: stain the sections with hematoxylin dye for 3 to 5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, return to blue with blue-returning solution, and rinse with running water. Then add 85% and 95% gradient alcohol for dehydration for 5 minutes each, and then add eosin staining solution for 5 minutes. Finally, add absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, absolute ethanol III for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes to make it transparent. The slides are sealed with neutral gum for microscopy and image acquisition and analysis.

Tissue damage was evaluated through pathological sections, and scar tissue was pathologically analyzed. Observe fibroblasts, capillaries, collagen fibers, etc. under a microscope and score them. The scoring criteria are:

1-3 points, no ~ minimal cell aggregation. No granulation tissue or epithelial transition;

Score 4-6, thin, immature granulation tissue, mainly inflammatory cells, with a small amount of fibroblasts, capillaries or collagen deposition. minimal epithelial cell migration;

Score 7-9, medium-thick granulation tissue, which is mainly composed of inflammatory cells and converted to fibroblasts and collagen deposition. Extensive neovascularization. Moderate degree of epithelialization and migration;

10-12 points, thick vascular granulation tissue, mainly fibroblasts and extensive collagen deposition. Epithelium partially or completely covers the wound surface. According to this standard, the 16 pathological slides were scored as shown in Table 16. The HE stained pathological sections of the scar areas on the ear surfaces of rabbits in four different groups 28 days after administration are shown in Figures 1 to 4 respectively.

Table 16. HE pathological section scores

Experimental results show that the pirfenidone topical preparation group (G1) of the present invention has the most obvious fiber repair effect, followed by the control prescription C1 (G3). There is no difference in the scores between the blank matrix group (G2) and the model group (G4). It shows that the pirfenidone topical preparation of the present invention has a very good therapeutic effect on scars, and the effect is better than the control prescription C1 (G3).

Test Example 5 Pharmacokinetic Properties Study

This test case aims to study a single topical application of pirfenidone topical preparation in mini pigs, detect the concentration of active ingredients in plasma, and evaluate the pharmacokinetics (PK) characteristics, systemic exposure and safety of mini pigs after topical administration. sex.

Experimental materials: male Bama Miniature Swine (3-5 months old, weight 9-10kg, purchased from Shanghai Jiaqian Biotechnology Co., Ltd.), experimental preparation (preparation prescription 6 of the present invention).

Experimental method: The day before the experiment, 2 minipigs were fasted overnight. On the day of the experiment, after weighing the body weight and calculating the administration area, select an area marked with the corresponding area on the back of the mini pig based on 10% of the body surface area, remove the hair, clean it, and draw the preparation at a dosage of 20 mg/ ^cm2 and apply it evenly. Cover the administration area with gauze and secure it with a vest.

Blood samples were collected at 0min before administration and at 15min, 30min, 1h, 2h, 3h, 4h, 6h, 8h, 10h and 24h after administration into K2EDTA anticoagulant tubes, stored on wet ice, and centrifuged within 1h. (1500~1600g, 4℃) for 10 minutes, separate the plasma, store the obtained plasma sample in an environment of -90~-60℃, and place it on dry ice for transportation.

The LC-MS/MS bioanalytical method was used to detect the drug concentration in the plasma of mini pigs. The non-compartmental model was used to analyze the blood drug concentration-time data using WinNonlinTM (Version8.3, Certara, USA) to evaluate its effect on mini pigs. In vivo pharmacokinetic properties. Table 17 shows the pharmacokinetic parameters of pirfenidone in the plasma of male minipigs after topical administration of a topical formulation of pirfenidone.

Table 17. Pharmacokinetic parameters of pirfenidone topical formulations


Note: NR means cannot be calculated.

The experimental results show that after topical application of pirfenidone topical preparation at a dose of 20 mg/cm ² , the maximum blood concentration (C _max ) in the plasma of male miniature pigs is 150 ng/mL, and the AUC (can be used to express exposure) is 2.695h*μg/mL, indicating that the pirfenidone topical preparation of the present invention has good long-term use safety.

Through the above embodiments and test examples, those skilled in the art can better understand the features and advantages of the present invention. However, the technical solution of the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc. made to the preparation formula, especially the co-solvent system, within the spirit and principles of the present invention, shall be included in the present invention. within the scope of protection.

Claims (10)

1. A topical pirfenidone preparation, characterized by comprising a therapeutically effective amount of pirfenidone, water and at least one co-solvent, the co-solvent being selected from the group consisting of dimethyl sulfoxide (DMSO) and alcoholic solvents.
2. The preparation of claim 1, wherein the alcoholic solvent is selected from one, two or more types of ethanol, propylene glycol, isopropyl alcohol, 1-propanol, and butylene glycol.

   Preferably, the co-solvent is a combination of DMSO and ethanol, or a combination of DMSO and propylene glycol, or a combination of DMSO, ethanol and propylene glycol.
3. The preparation of claim 2, wherein the amount of water in the preparation is 10 wt% to 55 wt%.
4. The preparation according to any one of claims 1 to 3, wherein when the co-solvent is a combination of DMSO and ethanol, based on the total weight of the topical preparation, the preparation contains 15wt% to 35wt% DMSO, 15wt% % to 30wt% ethanol; or

   When the co-solvent is a combination of DMSO, ethanol and propylene glycol, based on the total weight of the topical preparation, the preparation contains 15wt% to 35wt% DMSO, 15wt% to 30wt% ethanol, and 15wt% to 25wt% propylene glycol. .
5. The formulation of any one of claims 1-4, wherein pirfenidone is present in an amount of 1 to 14 wt%, based on the total weight of the pirfenidone topical formulation.
6. The preparation according to any one of claims 1 to 5, wherein the preparation further includes other pharmaceutically acceptable auxiliary materials, the auxiliary materials are selected from the group consisting of gelling agents, moisturizers, preservatives, antioxidants, and pH adjusters. , one, two or more of thickeners, stabilizers, chelating agents, oily materials, emulsifiers, penetration enhancers, antimicrobial agents, opacifiers, fragrances, colorants, surfactants.
7. The preparation of claim 1, wherein the dosage form of the preparation is selected from the group consisting of solutions, suspensions, creams, ointments, lotions and gels.
8. The preparation according to any one of claims 1-7, wherein the preparation contains 4wt% to 12wt% pirfenidone, 20wt% to 35wt% DMSO, 20wt% to 25wt% ethanol, 20wt% to 50wt% water, 0.3wt% to 1wt% gelling agent, 2wt% to 8wt% humectant, and an appropriate amount of pH adjuster adjust the pH range of the preparation to 5.0-7.5.
9. The preparation according to any one of claims 1 to 8, wherein the preparation contains 4wt% to 12wt% pirfenidone, 20wt% to 25wt% DMSO, 20wt% to 25wt% ethanol, 30wt% to 45wt% water, 0.3wt% to 1wt% gelling agent, 2wt% to 8wt% humectant, 0.05wt% to 1wt% preservative, and an appropriate amount of pH adjuster to adjust the pH range of the preparation to 5.0~ 7.5.
10. The use of the pirfenidone topical preparation according to any one of claims 1 to 9 in the preparation of medicines for preventing or treating diseases related to skin or connective tissue hyperproliferation and chronic skin damage.